Processes for extraction of omega-3 containing biomass oils from dried biomass

ABSTRACT

A method for using mechanical cartridges for the extraction of highly bioavailable omega-3 containing biomass oils from algal biomass is presented. Cartridges may contain an inner bore with an inlet and outlet, and may be amenable to adjustments in pressure and temperature, thereby permitting solvent-based extraction of algal biomass in which solvent temperature, pressure, retention time and flow rate are controllable.

REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 17/718,009, filed Apr. 11, 2022, which is a Continuation-in-partApplication of U.S. application Ser. No. 16/953,978 filed Nov. 20, 2020,both of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

BACKGROUND

Long chain Omega-3 oils are vital to human health and deficits in themcan have serious, negative health impacts. These range fromdevelopmental deficiencies in brain and neural networks to ourcardiovascular health, and more. Omega-3 oils are in high demand todaydue to supply limitations in aquaculture, fisheries, and other marinesources, at latest report, being able to supply only approximately 0.8million tons of Omega-3 fatty acids per year for human consumption. Thisis well below the current human nutritional demand of 1.4 million tonsrequired to supply the global population with 500 mg Omega-3 fatty acidsdaily and will be further exacerbated by population growth. Omega-3fatty acids deficiencies have been observed worldwide, and particularlyaffect populations located in North America, central Europe, the MiddleEast, India, Brazil, and the United Kingdom, with regional andsocioeconomic differences seen within these countries. There are threemajor Omega-3 fatty acids found in nature. Alpha linoleic acid (ALA),with 18 carbons and 3 double bonds, can be found, e.g., in flax seeds,soybean oil and olives. Docosahexaenoic acid (DHA) with 22 carbons and 6double bonds, and Eicosapentaenoic acid (EPA), with 20 carbons and 5double bonds, are only produced in aquatic species (algae) and can beextracted from krill or fish that eat the algae, or from the algaethemselves.

“Working algae”, i.e., algae grown using sunlight or artificial lightusing photosynthesis comprise mostly polar lipids, which fall into thecategories of glycolipids and phospholipids. By their nature, thesespecies have hydrophobic tails and a polar (hydrophilic) head—i.e., theyare essentially exotic natural detergents. These materials haveexceptionally high bioavailability, and thus, readily enter the humanbloodstream to deliver the above-mentioned health benefits. Various ofthese valuable materials have proven not found to be naturally producedin other sources, such sources may be of questionable sustainability, orhave proven to be difficult to feasibly access. Moreover, extractingthem from microalgae efficiently and in their pure, natural,bioavailable form, has not previously been effectively accomplished in acommercially successful manner, as existing commercial products havebeen hampered by an unattractive appearance, odor and texture. Whilehaving a higher bioavailability, algae extracts have traditionallyincluded components which give the extracted oil an overall, very dark,nearly black-ish, appearance, and a highly viscous feel, making themresemble an unattractive tar-like black solid, which remains relativelylow in concentrations of Omega-3 fatty acids.

Additionally, existing crude products have previously not been able tobe fully or adequately analyzed and characterized. Indeed, despitedecades of study, a fully characterized mass balance has not beenaccomplished or published without either reporting inclusions of highlevels (e.g., up to 10% or more) “unknowns” or “unidentified” materials,or by reporting results only “by difference” and putting suchessentially uncharacterized materials in a bucket labelled simply as“carbohydrates”, for example. This is especially undesirable in the caseof nutraceutical and pharmaceutical products.

Furthermore, existing crude products and the products refined therefromare typically generated via processes performed in standard reactors ormixing tanks. For high-throughput processing of algal biomass, theconstraints of standard reactors and mixing tanks can result inundesirably high costs and low production rates. Processes that refinenot just the separation science of algal extraction but the chemicalengineering that profits from it are needed in the art.

In summary, a process for producing a highly bioavailable, highconcentration Omega-3 containing, EPA-containing, high in polar lipids,especially glycolipids composition, which presents as a low viscosity,low chlorophyll content light amber to dark amber colored oil for use innutraceutical and pharmaceutical products, the contents of which arefully, or nearly fully characterized, is currently unavailable, yethighly desirable. Moreover, a method to obtain such highly desirablecompositions directly from plentiful, highly sustainable algae sources,would be ideal. Finally, a method is needed that satisfies the abovecriteria while being amenable to engineering solutions that permitrapid, lower-cost extraction of algal biomasses.

SUMMARY

The present disclosure describes a solution to the above problems byutilizing separation processes which include mechanical cartridges toextract oil extracts from, inter alia, microalgal biomass. The use of amechanical cartridge has been shown to reduce the processing time toone-third that required when using a standard reactor/mixing tank.Furthermore, the use of a mechanical cartridge has been shown to reducethe amount of solvent required to just one quarter that required whenusing the standard reactor/mixer. Mechanical cartridges of the presentsolution may comprise inner bores with an inlet and outlet to permitflow of solvent. Further, they may be heated, cooled, and pressurized,and may thereby control the pressure, temperature, retention time andflow rate of solvents that flow through them. The crude extract is thenfractionated into clean, well-characterized fractions, e.g., polarlipids, polysaccharides, and carotenoids, with high efficiency and veryhigh recovery. By virtue of the rapid cartridge-based extraction andinnovative fractionation process of the disclosure, a full mass balanceof the oil extract, and of the entire algal biomass, is now possible.

In one embodiment, the disclosed process includes a method forproduction of, e.g., a low chlorophyll content oil compositioncomprising the steps of obtaining a suitable algal biomass such as analgal paste or dried powder; extraction of the algal biomass with amechanical cartridge operable in pressurized, time-controlled settingsand a polar solvent such as an alcohol like ethanol to form an extractof algal lipids; extraction of the obtained extract with, e.g., anorganic solvent such as the hydrocarbons hexane or heptane, to separatethe fraction of non-polar lipids, transferring the, e.g., alcohol layercontaining pigments and polar lipids to a further stage of processing;adding water to the, e.g., alcohol layer extracted, e.g., with heptane,and then its sequential extraction with, e.g., heptane, to extract thepigment fraction and separating out the polar lipid fraction. Polarlipids can then be obtained from the fraction containing them byevaporation, and pigments can also be obtained by evaporation of thefraction containing them.

In an alternative embodiment, the disclosed process includes a methodfor production of a low chlorophyll content oil composition comprisingthe steps of obtaining an algal biomass which includes both polar andnon-polar lipid fractions and also has a chlorophyll concentration. Themethod further includes using polarity characteristics of the polar andnon-polar lipid fractions to segregate polar from non-polar componentsin the algal biomass, including substantially segregating thechlorophyll concentration with the non-polar lipid fraction. Additionalsteps include bleaching out substantially all the chlorophyllconcentration from the non-polar containing fraction; and re-combiningthe polar and non-polar lipid fractions to produce the lowchlorophyll-content LC-PUFA oil composition.

The above process embodiments produce an attractive composition for usein both the nutraceutical and pharmaceutical fields, particularly interms of reduced opacity and viscosity while maintaining a highbioavailability.

In a related embodiment, a method of fractionating and clarifying algalbiomass into its clean, precisely characterized components of LiquidExtracted Biomass, i.e., the residual post-extraction biomass (“LEA’),polar lipids, pure neutral lipids, chlorophyll, polysaccharides,carotenoids with high recovery of the total, and reporting a full algalbiomass balance, is provided. The method comprises the steps ofobtaining an algal biomass such as a paste or powder; extraction of thealgal biomass with a mechanical cartridge and a polar solvent such as analcohol like ethanol to form, e.g., an alcoholic extract of algallipids; extraction of the obtained, e.g., alcoholic extract with, e.g.,an organic solvent such as the hydrocarbons hexane or heptane, toseparate the fraction of non-polar lipids, transferring the, e.g.,alcohol layer containing pigments and polar lipids to a further stage ofprocessing; adding water to the, e.g., alcohol layer extracted, e.g.,with heptane, and then its sequential extraction with, e.g., heptane, toextract the pigment fraction and separating out the polar lipidfraction.

In an additional aspect, a method is provided for the extraction ofbiomass oils, which include, but are not limited, to lipids,chlorophyll, saccharides, carotenoids and Cannabidiols (CBD) from a dry,preferably powdered, biomass. The method includes the steps of obtaininga mechanical cartridge or extraction column with an inner bore and inletand outlet ends configured to permit solvent flow. The mechanicalcartridge is capable of being heated/cooled and pressurized and ofcontaining heated/cooled and pressurized solvent at ranges sufficient toseparate the target biomass oil from the dry biomass. For example, theoperating conditions permit the operator to melt lipids, thus allowingfor their extraction from the dry biomass in which they were contained.The mechanical cartridge itself is filled with dried biomass and themechanical cartridge can be heated/cooled to a predeterminedtemperature, typically between −30° C. and 150° C. Temperaturecontrolled solvent is pumped through the dry biomass filled mechanicalcartridge at a temperature, pressure, retention time and flow ratesufficient to remove the targeted biomass oils. For lipid containingbiomasses, the elevated temperature solvent melts the lipids in thelipids-containing portion of the dried biomass and thereby extractingthe lipids in liquid form from portions of the biomass retained in themechanical cartridge under the same conditions.

The disclosure further relates to a bioavailable, LC-PUFA-, Omega-3-,EPA-, polar lipids- and glycolipids/phospholipid-rich compositionespecially suitable for use in nutraceutical and pharmaceuticalcompositions, which can be considered as health or medical compositions,and as having other valuable end products. Starting material for thiscomposition, may be derived from algae as described herein. In oneembodiment, a composition is disclosed including a polar lipids fractionof a concentration of total lipids of at least 20% of the total lipidsby weight %; wherein the polar lipids fraction comprises at least 40%glycolipids by weight; and wherein the composition comprises no greaterthan 4% of its weight % as a chlorophyll concentration.

The composition, prepared as described above, with its total lipidshaving a fraction of at least 20 wt. % polar lipids, and its polarlipids having a fraction of at least 40 wt. % glycolipids, but thecomposition having less than a 4 wt. % chlorophyll fraction, may furtherinclude formulations with additive non-polar lipids and/or nutraceuticaloils such as DHA, Esters or salts of EPA or DHA (or mixtures thereof) orother beneficial additives as more fully described below, which alsoassist in providing for certain beneficial combinations of a morebioavailable, nutrient rich, lighter color, lower viscosity, oil.

These features and other features of the present disclosure will bediscussed in further detail in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses a schematic diagram of extraction process steps for anexemplary method of preparation of the oil of an embodiment of thedisclosure.

FIG. 2 is a photographic depiction of an embodiment of a composition ofthe disclosure made according to the exemplary method as set forth inFIG. 1 , having a desired target LC-PUFA-, polar lipid-, Omega-3-, EPA-and glycolipid-rich and chlorophyll-reduced concentration, withbioavailability, color and viscosity of a desired oil composition,embodiments of which are disclosed herein.

FIG. 3 is a photographic depiction of a powder obtained by an embodimentof a process of the disclosure made up substantially of non-lipidscomponents comprising, e.g., polysaccharides contained in the crudeethanolic algal extract made according to an embodiment of the processas described herein.

FIG. 4 is a graphical depiction of a spectral characterization of anembodiment of an ethanolic extract of Nannochloropsis and products afterpigments removal as discussed in Example 1. Y axis=AU (opticalAbsorption Units), X axis=nm (nanometers, wavelength).

FIG. 5 is a graphical depiction of a UV-Visible spectralcharacterization of an embodiment of an algal extraction as discussed inExample 2. Y axis=AU (optical Absorption Units), X axis=nm (nanometers,wavelength).

DETAILED DESCRIPTION

The following detailed description illustrates the claimed disclosure byway of example and not by way of limitation. This description willclearly enable one skilled in the art to make and use the claimeddisclosure, and describes several embodiments, adaptations, variations,alternatives, and uses of the claimed disclosure. Additionally, it is tobe understood that the claimed disclosure is not limited in itsapplication to the details and compositions specifically set forth inthe following description or illustrated by means of the figures. Theclaimed disclosure is capable of other embodiments and of beingpracticed or being carried out in various ways. Also, it is to beunderstood that the phraseology and terminology used herein is for thepurpose of description and should not be regarded as limiting.

As used herein, the term “lipids” means various organic compounds thatare insoluble in water. They include fats, waxes, oils, hormones andcertain components of membranes.

As used herein, the term “polar lipids” means amphiphilic lipids with ahydrophilic head and a hydrophobic tail. Examples of polar lipidsinclude phospholipids and glycolipids.

As used herein the term “non-polar lipids” means fatty molecules whereinthe charge distribution is largely evenly distributed, and the moleculesdo not have positively and negatively charged ends. Examples ofnon-polar lipids include triglycerides of the various fatty acids in theoil (e.g., EPA, palmitoleic acid and others, including mixtures thereofsuch as triglycerides (TAGs) comprising variations or combinations ofsuch fatty acids).

As used herein, the term “biomass” means the total mass of biologicalorganisms, including, e.g., plants and microorganisms, and can include,from a biological perspective, cellulose, lignin, lipids, sugars, andproteins. While aspects of the process of the invention are directlyrelevant to extraction, fractionation and clarification of algalbiomass, “biomass” should be considered to include other types oforganisms such as those of fungal or other origin, or “biomass” from agiven area or volume, unless expressly limited to algal biomass as thesole or primary source.

As used herein, the acronyms “EPA” and “DHA” refer to eicosapentaenoicacid and docosahexaenoic acid, respectively, as well as the salt andethyl ester forms of each compound. In its naturally-occurring acidform, EPA is a twenty-carbon unsaturated chain culminating in acarboxylic acid functional group. However, one of ordinary skill in theart appreciates that natural variants of this acidic form include analkaline salt, in which the deprotonated carboxylic acid is stabilizedby a counter anion, and an ethyl ester, in which two more carbons aresingly covalently bonded to the sp³-hybridized oxygen so as to result inan ester. In the case of the ethyl ester form, then, EPA has twenty-twocarbons. Similarly, DHA in its acidic form is a twenty-two-carbonunsaturated chain culminating in a carboxylic acid functional group andis understood to have natural variants including the alkaline salt andethyl ester form. In the ethyl ester form, consequently, DHA has twentyfour carbons. Alkaline salt forms of these compounds may manifestspontaneously as a result of particular chemical environments in whichthey are present. The transformation to the ethyl ester variants issimilarly facile, and these variants are also used as medicants totreat, for example, high blood triglyceride levels. Any use of theacronyms “EPA” and “DHA” in the present application should not beconstrued to exclude the alkaline salt or ethyl ester variants of eithercompound unless their exclusion is made explicit.

To produce the disclosed embodiments of clarified compositions of analgal biomass, an algal paste, which presents as a dark green or evenblack, highly viscous oil can be obtained using standard steps know bythose of ordinary skill in the industry. See, e.g., the production ofthe algal paste and useful varieties of algae employable, as describedin U.S. Pat. No. 8,591,912 B1 (hereinafter “Kadam and Goodall”),incorporated herein by reference and additionally discussed herein.Additionally, such an algal biomass useful for extraction may be dry andpresented in the form of an algal powder or other suitable form.

Procedures for obtaining the algal biomass extract, and starting algaeand extraction procedures for preparing the algal biomass can includethe following steps, as part of an extraction:

Obtain or prepare a biomass such as an algal paste or powder, e.g.,prepared from the algal paste from an appropriate species suitable forproducing such a target biomass. For example, and without limitation,dry algal powder can be prepared from an algae paste by, e.g., a drumdryer, a powder dryer, a refractance window dryer, a freeze dryer, anoven, etc. by procedures known to those skilled in such drying arts. Ingeneral, microalgae can be harvested by conventional means (including,but not limited to filtration, air flotation and centrifugation) and thealgal paste generated by concentrating the harvested microalgae to thedesired weight % of solids. In certain embodiments, the microalgae usedwith the methods of the invention are members of one of the followingdivisions: Chlorophyta, Cyanophyta (Cyanobacteria), andHeterokontophyta. In certain embodiments, the microalgae used with themethods of the invention are members of one of the following classes:Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. In certainembodiments, the microalgae used with the methods of the invention aremembers of one of the following genera: Nannochloropsis, Chiarella,Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium,Spirulina, Amphora, Trachydiscus and Ochromonas. Non-limiting examplesof microalgae species that can be used with the methods of the presentinvention include: Achnanthes orientalis, Agmenellum spp., Amphiprorahyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphoracoffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphoracoffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissimavar. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Bracteococcus minor, Bracteococcusmedionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri,Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomasperigranulata, Chlorella anitrata, Chlorella antarctica, Chlorellaaureoviridis, Chlorella candida, Chlorella capsulate, Chlorelladesiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca,Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorellainfusionum, Chlorella infusionum var. actophila, Chlorella infusionumvar. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorellaluteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorellaluteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima,Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorellaparva, Chlorella photophila, Chlorella pringsheimii, Chlorellaprotothecoides, Chlorella protothecoides var. acidicola, Chlorellaregularis, Chlorella regularis var. minima, Chlorella regularis var.umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorellasaccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex,Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorellastigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorellavulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorellavulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorellavulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo.viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorellatrebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcumsp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp.,Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotellameneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil,Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime,Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliellaprimolecta, Dunaliella salina, Dunaliella terricola, Dunaliellatertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaeraviridis, Eremosphaera sp., Effipsoidon sp., Euglena spp., Franceia sp.,Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnionsp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff. galbana,Isochrysis galbana, Lepocinclis, Micractinium, Micractinium,Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.,Nitschia communis, Nitzschia alexandrina, Nitzschia closterium,Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschiahantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschiamicrocephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschiapusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonassp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatorialimnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorellakessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum,Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae,Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii,Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis,Prototheca Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys,Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus,Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp.,Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula,Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosiraweissflogii, Trachydiscus and Viridiella fridericiana.

Preferred algal species are those, e.g., rich in LC-PUFA oil sources.Exemplary of such algae strains are strains from Nannochloropsis,Chlorella or Trachydiscus minutus algal varieties.

The algal paste or other suitable form of algal biomass can be processedas follows: extract, e.g. with a polar solvent such as an alcohol,including, ethanol to form, e.g., an alcoholic extract of algal lipidswith a low water content (forming, e.g., an ethanolic extract ofNannochloropsis lipids (hereinafter referred to for brevity as “EEN”);extract the obtained EEN with, e.g., an organic solvent such as thehydrocarbons hexane or heptane to separate out the fraction of non-polarlipids (e.g., triglycerides, waxes, carotenes), thus forming a“non-polar lipid fraction” (F#1 of FIG. 1 ) in the heptane layer. Thealcohol layer containing pigments and polar lipids can be transferred toa further stage of processing. See FIG. 1 . Other solvents such ascarbon dioxide or carbon dioxide ethanol mixes may be considered.

The next stage of processing can involve adding water to the polarsolvent layer extracted, e.g., with heptane and then its sequentialextraction with, e.g., heptane, to extract the pigment fraction (seeFIG. 1 , heptane layer, F #3) and separating out the polar lipidfraction (see FIG. 1 , water-alcohol layer, F #2). Polar lipids can beobtained from the F #2 fraction by evaporation, and pigments can beobtained by evaporation of the F #3 fraction. The F #1 layer may containan amount of chlorophylls and carotenoids, which can be removed bymethods known to those skilled in the art, e.g., methods known for theproduction of edible oils. Examples of these methods includeadsorption-filtration using silica gel, bleaching clays such as B80,T41, activated carbon, and others. As a result of the selective removalof chlorophyll from the F #1 fraction, in this embodiment, a largelyclear to dark amber, including reddish, somewhat liquid to semi-solidoil can be obtained.

In an alternative embodiment, the method for production of a lowchlorophyll content oil composition, via the extraction of algal biomasssuch as algal paste or powder, so obtained includes both polar andnon-polar lipid fractions and also has a chlorophyll concentration.Polarity characteristics of the polar and non-polar lipid fractions ofthe biomass are used to segregate polar from non-polar components in thealgal biomass, including substantially segregating the chlorophyllconcentration with the non-polar lipid fraction. As illustrated inExample 2 below, additional steps include bleaching out substantiallyall the chlorophyll concentration from the non-polar containingfraction; and re-combining the polar and non-polar lipid fractions toproduce the low chlorophyll-content oil composition.

In addition to the low chlorophyll-content aspect of the composition,the present invention also provides for isolated oil derived from algaewhich are rich in LC-PUFAs, including at least one Omega-3 fatty acidsuch as, but not limited to, EPA or DHA, at least partially in the formof whole and unhydrolyzed phospholipids and whole and unhydrolyzedglycolipids extracted by the above processes. The oil produced by suchprocess is unexpectedly high in polar lipids such as phospholipids andglycolipids. It is known that krill oil, by virtue of the containedphospholipids (a polar lipid) has higher bioavailability in mammals thandoes fish oil, which comprises almost exclusively neutral lipids(triglycerides). See, Jan Philipp Schuchardt et al., Lipids in Healthand Disease 2011, 10:145. Remarkably, the oils extracted from algae suchas Nannochloropsis using the inventive methods showed bioavailability tomammals that surpassed even krill oil.

In another embodiment of the lipid-extraction aspect of the invention,the inventors have created a process for the extraction of biomass oils,which include, but are not limited to lipids, chlorophyll, saccharides,carotenoids and CBD utilizing a heated (or in certain circumstances,cooled), pressurized mechanical cartridge or extraction column toextract, e.g., in the case of lipids, by melting them fromlipid-containing dry, preferably powdered biomasses (see above forvarious methods of producing powdered biomasses). Such lipid-containingpowdered biomasses to which this aspect applies include, but are notlimited to, algal biomasses, such as the preferred sources (autotrophic,Nannochloropsis-derived, etc. algal biomasses described elsewhere inthis application).

The biomass oil, e.g., lipid extraction, using a pressurized,temperature-controlled mechanical cartridge to, e.g., melt the lipidscontained in a dried biomass, has a number of advantages. For instance,its use provides for a significant reduction in the time required forthe lipid extraction to reach completion. The use of the mechanicalcartridge has been shown to reduce the processing time to one-third thatrequired when using a standard reactor/mixing tank. For another, thisprocess allows for a substantial reduction in the solvent required forthe extraction. The use of the mechanical cartridge has been shown toreduce the amount of solvent required to one-fourth that required whenusing the standard reactor/mixer

The cartridge or extraction column selected is dimensioned to have aninner bore and inlet and outlet ends configured to permit containment ofthe dry biomass and also to permit and regulate solvent flow. The innerdiameter of the mechanical cartridge is constructed of a material,preferably stainless steel, capable of being heated and pressurized andof containing heated and pressurized solvent at ranges sufficient tomelt and separate out lipids (or otherwise extract out a target oilbased on differentials in reaction to temperature and pressure). Theinner diameter of the cartridge can preferably be between 25 and 1000 mmand a straight length, not including the inlet and outlet connections,of between 100 to 1000 mm. More preferably, these can be between 300 and500 and 150 mm to 300 mm, respectively. Even more preferably these are400 mm and 200 mm respectively. The inlet and outlet ends of themechanical cartridge, which provide for and regulate solvent flowthrough the inside of the cartridge and through the powdered biomasscontained within, include grates, screens, gates, etc. which allow forcontrol and regulation of rates of flow and retention time for thesolvent. They also can be used to limit the leftover solid material frompassing out of the mechanical cartridge.

The material, and its physical form, chosen to fill the cartridge willdepend to a degree on the target biomass. For algal biomass as a target,the cartridge can be filled with a lipid-containing portion of a dried,preferably powdered, biomass and is heated to a predeterminedtemperature to facilitate the lipid melting. Non-limiting examples ofways to heat the cartridge itself include jacketing the cartridge,passing a heating fluid around its exterior, immersing it in a heatingsolution, and via electrical resistance or induction.

In this embodiment, after the cartridge is filled with alipid-containing portion of a powdered biomass, preheated solvent ispumped through the cartridge at a temperature, pressure, retention timeand flow rate optimized to melt the lipids in the lipids-containingportion of the powdered biomass, thereby extracting the lipids in liquidform from portions, e.g., chlorophyll-containing content, retained inthe cartridge under the same conditions.

The solvent used in the cartridge method may be any applicable solvent.Non-limiting examples include alcohols such as ethanol, hexane, heptane,carbon dioxide or solvent mixtures. Moreover, the method is applicableto a full range of lipid profile, i.e., polar to non-polar for theassociated crude extraction from a dried biomass.

An operating principle for the use of the mechanical cartridge is itsallowance of extraction with solvents at targeted, e.g., for algalbiomasses, elevated temperatures, and pressures. Preferred temperaturesfor the solvents can be in a range between −30 degrees C. and 150degrees C. For lipid extraction, more preferably between 70 degrees C.and 110 degrees C., and even more preferably 90 degrees C. Preferredoperating pressure of the solvent is between 5 bar and 100 bar, morepreferably between 25 and 50 bar, and even more preferably is 30 bar. Asnoted, depending on the material to be extracted, the solvent used, andthe biomass from which the target material is being extracted, even acooled solution could similarly be employed for suitable biomassmaterial extraction in which a cooled solvent is more advantageous.

Such mechanical cartridge lipid extractions are readily combined withfurther processing, e.g., via the approaches presented in Examples 1 and2 below.

EXAMPLES

The following non-limiting examples are illustrative of the invention,both for clarifying and determining specific components of the oilcomposition, and for producing the LC-PUFA-rich and low-chlorophyll,low-polysaccharide-containing oils as further described herein:

Example 1 2.1 Process Protocol for this Example (as Referred to in FIG.1)

1) 100 g of Nannochloropsis algal paste (22-27% of solids in water) wasweighed out. (Stage 1, FIG. 1 )

2) The algae paste was placed in a 2 L flask and 850 ml of alcohol wasadded. (Stage 1, FIG. 1 )

3) The algae was extracted for 45 min at a temperature of 70° C. withvigorous stirring. (Stage 1, FIG. 1 )

4) Solid algae residues were filtered out from the ethanol extract(vacuum filtration). (Stage 2, FIG. 1 )

5) The ethanol extract from the previous stage was placed into aseparatory funnel (2 L), 300 mL of heptane was added to the resultingextract, stirred vigorously for 2 min, the layers were separated, thetop layer was carefully selected and placed in a separate flask andabout 120 mL of a green heptane layer was obtained. (Stage 3, FIG. 1 )

6) Another 100 mL of heptane was added to the ethanol layer, stirredvigorously for 2 min, the layers were separated (ethanolic layer—lower,and heptane layer—upper), the top layer was carefully selected andcombined with the heptane layer obtained in the previous stage (˜200-220mL of combined green heptane layers was obtained, fraction F #1). (Stage4, FIG. 1 )

7) 1 gram of silica gel was added to the entire heptane layer obtainedand, after vigorous stirring for 5 minutes, the slurry was filteredthrough a layer of one gram of silica gel (instead of silica gel,activated carbon or T41 bleaching clay can be used). Due to variationsin the properties of various silica gels, activated carbons, andbleaching clays, the actual amounts of the materials should be adjustedon plant. (Stage 8, FIG. 1 )

8) The lower (ethanolic) layer was taken from this protocol stage 6, 350mL of water and 200 mL of heptane were added, and the mixture was shakenintensively for 2 mins. After about 5 min of settling the separatephases, the top layer was carefully selected and placed in a separateflask (˜400 mL of a green heptane layer was obtained at this stage).(Stage 5, FIG. 1 )

9) The lower layer was repeatedly extracted from the above stage with200 mL of heptane (3×200 mL). The top layers were carefully selected andcombined with the heptane layer obtained in the previous stage.(˜1000-1020 mL of combined green heptane layers was obtained, fraction F#3). (Stage 5, FIG. 1 )

10) The extracted lower layer contained clarified polar lipids (F #2).This fraction can be polished by 3 g of, e.g., Amaze-N bleachingsorbent, from Helix Chromatography (15 E. Palantine Rd. #118, ProspectHeights, Ill. 60070; helixchrom.com), (or similar sorbents can be used,if needed).

11) The fractions obtained were evaporated in vacuum with heating <45°C.

The extraction process exemplified above is fundamentally presented as abatch process. However, an automated process making use of appropriatecontrols and/or liquid-liquid centrifuges to accomplish the protocolsdescribed could similarly by employed by those skilled in this art.Automation of all of the examples/embodiments presented is possibleusing equipment known to people knowledgeable in the art.

The above process steps and experimental results demonstrate a highlyefficient exemplary method for a liquid-liquid extraction, removingchlorophyll and a fraction of carotenoids from phototrophic(autotrophic) algal extracts, such as for Nannochloropsis or Chlorellalipids. By use of the above embodiment of a method of the disclosure,more than 99% of chlorophylls α and β and pheophytins were removed froman ethanolic extract of Nannochloropsis (EEN), as well as abouttwo-thirds of the carotenoids (medium polarity carotenoids). In doingso, at least 90 weight %, preferably greater than 95 weight %, morepreferably greater that 97.5 weight %, even more preferably greater than98.0 weight %, and most preferably, greater than 99 weight % of theoriginal mass balance is fully retained and can be characterized as toits principal component parts without resorting to leaving a largeportion of the mass balance as simply being uncharacterized orcharacterized only “by subtraction” or “by difference”. See Table 1:Extraction mass balance (composition) and Table 2: Principal componentsof the ethanolic extract of Nannochloropsis for mass and weight %analysis of the compositions. (Representing 99.86% of the total algalbiomass by weight %.) See FIG. 4 for a spectral characterization of theNannochloropsis ethanolic extract after removal of pigments.

TABLE 1 Extraction mass balance (composition) Extraction Mass BalanceItem Value Algal paste concentration, % 27.5% Initial paste mass, g100.0 g Solid mass (recalculated), g  27.5 g Extract volume, mL 950Extract mass, g 9.97 Concentration (mg/mL) 10.5 Extract mass, % 36.25Residue mass, % 63.75

TABLE 2 Principal components of the ethanolic extract of NannochloropsisPrincipal groups of components Item Mass, g Weight % Non-polar lipids1.533 15.36 Medium polarity lipids 2.605 26.09 Chlorophyll total 0.6976.98 Carotenoids total 0.223 2.23 Polar lipids 3.62 36.23 Sugars andpolar components 1.294 12.97 Total: 9.951 99.86

While viscosity measurements can vary to a degree depending on suchfactors as temperature, compositional concentrations of variousingredients in a formulation, etc., a viscosity reading taken for anembodiment of a Nannochloropsis extract prepared as described aftercombination of a polar lipids fraction with a neutral lipids fraction at25° C. as described herein was noted to be @ 165,000 mPa··'s.

In summary, the products shown in Example 1 can be broken down intothree principal fractions of the incoming ethanolic extract ofNannochloropsis (EEN)—1) Fraction (F #1), non-polar lipids, mainlytriglycerides; 2) Fraction (F #2), polar lipids, including glycolipidsand phospholipids; and Fraction (F #3), a fraction of medium polarity,comprising di- and mono-glycerides, free fatty acids (FFA's),carotenoids and chlorophyll. The clarified F #1 and F #2 fractions canbe used as sources of valuable lipids high in palmitoleic andeicosapentaenoic acids (EPA). F #3, as a concentrate of naturalpigments, including chlorophyll, astaxanthin, zeaxanthin, and others,also have value as food additives. Fractions F #1-3 can each be used asfood additives and are valuable raw materials with high biologicalpotential.

EXAMPLE 2

A sample of a dark green paste of the algal biomass was preparedgenerally in accordance with a method outlined in U.S. Pat. No.8,591,912 B1 (see, generally, Col. 6, line 62 to Col. 9, line 3) anddiscussed herein (see, e.g., at [0025]. The algal biomass paste wasextracted with hot absolute ethanol. Specifically, 66 g algal paste,3×250 mL ethanol, at 75° C., 30′ while stirring at 500 rpm each,centrifugal separation at 4450 rpm for 10 minutes, yielded a specimenalgal extract.

An analysis conducted of the oil extract demonstrated the principalpolar lipids in the algae specimen to be: 1) glycolipids (monogalactosyldiglycerides (MGDG) and digalactosyl diglycerides (DGDG) and 2)phospholipids (phosphatidlycholine, phosphatidylethanolamine, andphosphatidylinositol) (See Table 3, herein.)

TABLE 3 Mass, Weight, Item g % Group Sample mass (wet algae paste) 66.1n/a Quantification Dry algae (calculated) 15.87 100 Quantification Dry*residue after extraction 9.32 58.7 Quantification Crude extract mass6.56 41.3 Quantification Non-Lipid Components 1.95 12.3 ComponentsNon-polar lipids** 1.11 7 Lipids Glycolipids*** 1.76 11.1 LipidsPhospholipids**** 0.78 4.9 Lipids Chlorophyll 0.75 4.74 PigmentsCarotenoids 0.21 1.33 Pigments *permanent weight on drying at roomtemperature (final moisture was not tested) **TAG, DAG, FFA,Phytosterols ***Glycolipids (AMGDG, MGDG, DGDG, SQDG) ****Phospholipids(PC, PE, PI, PA, PG)

An embodiment of a bioavailable, low chlorophyll content, polarlipids-rich, LC-PUFA-rich, Omega-3 oil rich oil of the disclosure asquantified herein was prepared from the above starting material usingthe following additional steps:

1) Polar lipids were separated from a mixture of non-polar lipids,chlorophylls, and other components based on differences in polarity.

2) Chlorophylls were bleached from the remaining non-polar lipidcomponents using well developed protocols for vegetable oil bleachinggenerally as described in Example 1 above; and e.g. 1 gram of silica gelwas added to the entire heptane layer obtained and, after vigorousstirring for 5 minutes, the slurry was filtered through a layer of onegram of silica gel (instead of silica gel, activated carbon or T41bleaching clay can be used). Due to variations in the properties ofvarious silica gels, activated carbons, and bleaching clays, the actualamounts of the materials should generally be adjusted on plant.

3) The polar lipids fraction of (1) above was combined with the bleachednon-polar lipids of (2) above.

A bioavailable polar lipid-rich, low chlorophyll-containing oilcomposition having a generally low viscosity and with a nearly clear tolight brown color was obtained. See FIG. 5 for spectral analysis. Thecomposition was a waxy solid at ambient temperature of @ 70 degreesFahrenheit. The composition melts when warmed and exhibits low viscositywhen blended with other oils such as triglycerides and the like.

EXAMPLE 3

Use of an extraction column/mechanical cartridge. 1) 58.85 g of dryalgal powder of Nannochloropsis (water content 1.2%, Karl Fishier) wereplaced into a stainless-steel cartridge (ID 25 mm, L 150 mm, V 75 cm3).

2) The thermostated extraction column filled with the algal powder waskept at 90 degrees C. during the extraction.

3) Dry ethanol (0.1% (w/w) water, KF) at 90 degrees C. (preheated in aheat exchanger), was pumped through the extraction column at a flow rateof 4.0 ml/min (0.8 ml/min/cm2), total pumped volume 210 ml. he eluate(extract) appearing as a dark green liquid was collected. Totalextracted mass was 15.91 g, dry residue weight (extracted algae), 42.85g. Mass % of the extract was 27.1%.

Once the lipids portion has been separated from the chlorophyllcontaining fraction of the biomass, i.e., the chlorophyll containingfraction has been removed or substantially reduced in the lipidsportion, as shown by the process as exemplified above, the resultingextract that is now low in, or devoid of, chlorophyll, can be furtherfractionated using, e.g., liquid-liquid protocols as described inExamples 1 and 2 to afford clean fractions of the other components suchas the polar lipids, non-polar lipids, etc.

An analysis of the oil composition of embodiments of the bioavailableoil of the disclosure made using the above processes described hereindemonstrated that oils having the following components and features(column 1), and component ranges (column 2), as set forth in Table 4were obtained from algal biomass:

TABLE 4 Components/ Ranges of Features Components of Exemplary ofDisclosed Composition Compositions Total Lipid concentration inthe >90% >75% oil product Polar Lipid fraction of lipids −70% >20% TotalOmega 3 content in oil −30% >20% product Total EPA content in the oil−30% >20% product Glycolipid concentration as % of −60% >30% polar lipidGlycolipid as % of oil product −40% >20% Phospholipid as % of polarlipid −40% >20% Phospholipid as % of oil product −25% >10% Totalchlorophyll concentration <0.1%   <4% in oil product Totalpolysaccharide content  <1%  <4% (%) in oil product Color/Capacity? DarkAmber - Dark Amber - Clear Clear

Modest variations in the weight % of components and othercharacteristics of the oil disclosed in this application may be obtainedby alterations to the process employed, as is known to those skilled inthis art. However, preferably the weight % of the polar lipid fractionof the total lipid concentration of the produced oil exceeds 20%,preferably exceeds 30%, more preferably exceeds 40%, even morepreferably exceeds 50%, and still more preferably is about 70% or above.Also, preferably, the weight % of the chlorophyll concentration in theoil product is less than 4% of the weight of the total oil product, morepreferably, it is less than 3.0%, yet more preferably, it is less than2.0%, even more preferably, it is less than 1.0%, 0.75%, 0.50%, 0.2% andstill more preferably, it is 0.1% or below. Also, preferably, the weight% of the polysaccharides concentration in the oil product is about 4% orless of the weight of the total oil product, more preferably, it is lessthan 3.0%, yet more preferably, it is less than 2.5%, 2.0%, even morepreferably, it is less than 1.0%, 0.5% or below. Additionally, theweight % of glycolipids as a weight % of total polar lipids exceeds 20%,preferably exceeds 30%, more preferably exceeds about 40%, even morepreferably exceeds 50%, 60%, 70% and still more preferably it is 80% orabove, and its weight % of the total oil composition exceeds 10%, morepreferably 20%, and still more preferably is about 25% or above.Additionally, the weight % of phospholipids as a weight % of polarlipids exceeds 20%, more preferably exceeds 30%, and still morepreferably exceeds 35%, and their weight % of the total oil compositionexceeds 20%, more preferably 30%, and still more preferably 40%. Whilenot being bound by any particular theory, applicant believes that by thecombination of these characteristics, including very low chlorophyllconcentration in the oil product, an attractive, lightly colored, nearlyclear, to amber, up through darker amber color is produced, and in apreferred embodiment, nearly clear to amber. Also, EPA concentration ofthe total oil product content is at least 20 weight %, more preferablyat least 25%, and still more preferably at least about 30 weight % orgreater is produced. Similarly, the Omega-3 content of the oil productis at least 20 weight %, more preferably at least 25 weight %, and stillmore preferably at least 30, 40 weight % or greater is produced.Considered as a whole, the weight % of LC-PUFA content of the oilproduct at least 20%, at least 25%, 30%, 40% and 50%.

Elevated bioavailability of the resultant oil is also achieved.Moreover, unlike some prior art methods of extracting and fractionatingmicroalgae, the disclosed method does not involve aggressive chemistrysuch as the use, e.g., of strong mineral acids which frequently destroypolar lipids and can significantly degrade other valuable fractions ofthe algal biomass.

Also obtained in embodiments of processes of the disclosure are powdersof non-lipids components such as polysaccharides of the crude ethanolicalgal extracts. See FIG. 3 .

It should also be noted that for LC-PUFA, Omega-3, EPA-containing, highpolar content oils of the disclosure, there is a greatly reduced weight% of the following chlorophylls: 1) chlorophyll a, 2) protochlorophylla, and 3) methylchlorophyllide. Preferably, the weight % of thechlorophylls of the composition is less than 4%, preferably less than3%, yet more preferably less than 2%, even more preferably less than 1%,still more preferably less than 0.5%, 0.5%, and still even morepreferably less than 0.1% of the total weight % of the composition.

In another embodiment of a composition of the disclosure, thecomposition has an enhanced weight % of several other ingredients,including carotenoids, e.g., carotenes (α and β), and zeaxanthins(yellow pigments), and canthaxanthin and zeaxanthin (reddish pigments),with total carotenoids making up more than 0.5% of the total weight ofthe extract and preferably more than 1%.

In other alternative embodiments of the disclosure, the bioavailable,high polar-lipids containing, EPA-containing, low chlorophyll contentoil compositions, embodiments of which are disclosed herein, can bepresented as formulations in which other useful ingredients are added.These other useful ingredients can be added alone, or in one or morecombinations, e.g., combinations with other essential oils, dietarysupplements, health supplements and the like. Specific examples include,but are not limited to—1) other omega 3 containing oils or componentssuch as DHA and EPA (e.g., in the form of the neutral lipids extractedas a product in the instant invention or externally sourced), thelysolipids from the instant invention, or externally sourced, ethylesters of DHA or EPA; 2) antioxidants such as carotenoids, includingastaxanthin, lutein, zeaxanthin, lycopene, carotenes (alpha and beta),cryptoxanthin, and mixture thereof (including the carotenoid fraction ofthe instant invention); 3) vitamins, such as vitamin C and D; 4)cannabinoids, such as cannabidiol (CBD), and 5) other combinations. Itis understood that such formulations including some of these specieswith less-colored and/or less-viscous compositional profiles, may reducethe overall color profile and viscosity of formulations which includethe fatty acid compositions with high polar lipid, high glycolipid, andlow chlorophyll concentrations prepared by embodiments of the processesdisclosed herein. Thus, this may be achieved, e.g., by inclusion ofnon-polar lipids, either added back in from original biomass stock, orfrom an external source, or by preparation of formulations whichdemonstrate such attributes.

Examples of nutraceutical formulations including blends of the polar EPAfraction described above with DHA (Omega-3) can beneficially be in aratio from 10-90 to 90-10, wherein preferable levels of the polar EPAformulation component being mixed at 20-50%. Uses for such formulationsinclude both use as a key food supplement/nutraceutical in its own rightfor cardiovascular health, mood, anti-depression and more, and also as adelivery system for other neutral lipids and components it is formulatedwith. This can be DHA, other neutral forms of EPA, or mixtures thereof.Astaxanthin at levels of 0.04% to 10%, preferably 0.1% to 2%, and morepreferably 0.2% to 1% can also be beneficially formulated, either withpure polar EPA lipids or blends thereof with neutral EPA and/or EPA.Another component that could be beneficially added to such a formulationis coenzyme Q10 at levels of around 1-50%, preferably about 2-20% on thepolar EPA, either pure or in any of the above formulations.

In addition to other attributes, formulation blends with added neutrallipids, e.g., can be useful to target various viscosity levels, such as50,000 cps, preferably less than 10,000 cps, more preferably less thanabout 2,000 cps, and most preferably, about 300 cps or less.

In view of the above, it will be seen that the several objects andadvantages of the present disclosure have been achieved and otheradvantageous results have been obtained.

As various changes could be made in the above constructions withoutdeparting from the scope of the disclosure, it is intended that allmatter contained in the above description or shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

1. A method for extraction of a biomass oil, which may include lipids,from a dried biomass, comprising: 1) obtaining a mechanical cartridgewith an inner bore and inlet and outlet ends permitting solvent flow,capable of being temperature controlled and pressurized and ofcontaining temperature controlled and pressurized solvent at rangessufficient to extract a target biomass oil, such as to melt lipids; 2)filling the mechanical cartridge with a target biomass oil-containingportion of the biomass; 3) if required for targeted extraction, heatingor cooling the mechanical cartridge to a predetermined temperaturerange; and, 4) pumping a solvent through the biomass-filled mechanicalcartridge at a temperature, pressure, retention time and flow ratesufficient to extract the target biomass oil in the target biomassoil-containing portion of the biomass and thereby extracting the targetbiomass oil from portions of the biomass retained in the mechanicalcartridge under the same conditions.
 2. The method of claim 1 whereinthe mechanical cartridge has an inner diameter of between 25 and 1000 mmand a straight length, not including inlet and outlet connections of 100to 1000 mm.
 3. The method of claim 2 wherein the mechanical cartridgehas an inner diameter of between 300 and 500 mm and a straight length,not including inlet and outlet connections of 700 to 300 mm.
 4. Themethod of claim 1 wherein the biomass comprises an algal biomass.
 5. Themethod of claim 4 wherein the biomass comprises an autotrophic algalbiomass.
 6. The method of claim 5 wherein the biomass comprises aNannochloropsis, Trachydiscus or Chlorella biomass.
 7. The method ofclaim 1 wherein the inlet and outlet ends of the mechanical cartridgecomprise grates, screens or gates configured to provide flowdistribution of the solvent through the biomass and limit solid materialfrom passing out of the mechanical cartridge.
 8. The method of claim 1wherein the temperature control comprises heating of the mechanicalcartridge and the method of heating is selected from the groupconsisting of one or more of jacketing, passing a heating fluid aroundthe exterior of the mechanical cartridge, immersion in a heatingsolution, by electric resistance and by induction.
 9. The method ofclaim 1 wherein the solvent comprises an alcohol, hexane, heptane orcarbon dioxide.
 10. The method of claim 1 wherein the targeted biomassoil comprises a polar lipid.
 11. The method of claim 1 wherein thetargeted biomass oil comprises a non-polar lipid.
 12. The method ofclaim 1 wherein the solvent is heated to a range between 40 degrees C.and 150 degrees C. during extraction of the lipid.
 13. The method ofclaim 1 wherein the solvent is heated to a range between 70 degrees C.and 110 degrees C. during extraction of the lipid.
 14. The method ofclaim 1 where operating pressure of the solvent is between 5 bar and 100bar.
 15. The method of claim 1 where operating pressure of the solventis between 25 bar and 35 bar.
 16. The method of claim 1 wherein thetargeted biomass oil is a lipid, and the lipid-containing portion of thebiomass is subjected as the extracted lipid to further processingsubsequent to the steps of claim
 1. 17. The method of claim 16 whereinthe further processing subsequent to the steps of claim 1 comprisefractionation between polar and non-polar lipids.
 18. The method ofclaim 16 wherein the further processing subsequent to the steps of claim1 comprise liquid-to-liquid fractionation.
 19. The method of claim 17wherein the further processing comprises the steps of: a. Where theextracted biomass comprises an algal biomass, sequestering the algalbiomass; b. Extracting the algal biomass with a polar solvent to form apolar extract of algal lipids with a low water content; c. Furtherextracting the polar extract with an organic solvent to separate out afraction of non-polar lipids, thus substantially removing a non-polarlipids fraction in an organic solvent layer; d. separating out a polarlayer containing pigments and polar lipids; e. adding water to the polarlayer and further extracting with sequential additions of the organicsolvent, whereby a pigment fraction is substantially removed to form awater-polar layer containing the polar lipids; and, f. separating out apolar lipids fraction from the water-polar layer by evaporation.
 20. Themethod of claim 16 wherein the further processing comprises the stepsof: a. Where the extracted biomass comprises an algal biomass,sequestering the algal biomass whereby an algal oil or extractcomprising both polar and non-polar lipid fractions, and having at leastsome chlorophyll concentration; is obtained b. using polaritycharacteristics of the polar and non-polar lipid fractions to segregatepolar from non-polar components in the algal oil or extract; c. removingsubstantially all of the at least some chlorophyll concentration fromthe non-polar containing fraction; and d. re-combining the polar andnon-polar lipid fractions to produce a low to substantially one lackingany chlorophyll content oil composition.